Simplot Tutorial: Virus Recombination Analysis

1.Introduction

Gene recombination is an important mechanism in the evolution of viruses, allowing for rapid genetic variation. To reveal the role of genetic mechanisms such as recombination in certain gene evolutions, recombination analysis is necessary to identify potential recombination signals and assess their reliability. This tutorial will guide beginners through the use of Simplot, a popular tool for virus recombination analysis.

1.1 What is Simplot?

Simplot, short for Similarity Plotting, is a widely-used tool for recombination analysis. It has been cited in over 900 publications, indicating its popularity. However, it only supports Windows systems and has not been updated since version 3.5.1 in 2003.

1.2 How Simplot Works

Simplot analyzes sequences by plotting their similarities against reference sequences. The resulting plots can indicate the presence of recombination signals:

1. If the reference sequences' plots are approximately parallel lines, it suggests high sequence similarity with one of the reference sequences and no recombination signal.
2. If the reference sequences' plots cross, it suggests the possibility of a recombination signal.

2.Steps

Step 0: Download Simplot

download_Simplot

Step 1: Sequence Alignment

1. Group and align sequences. It is recommended to use unique names for different reference sequence groups to facilitate automatic grouping by Simplot.
2. Save the alignment as a .fasta file.

Step 2: Running Simplot

1. Launch Simplot and open SeqPage. Click on "File" -> "Open" and select the saved .fasta file. All sequences will be selected by default.
2. Change the way groups are identified 18
3. Adjusting parameters :Window Size ,Step Size,Vertical Axis
4. Switch to the "Simplot" tab and click on "Command" -> "Query" and select Human_coronavirus_229E
5. The "Start Scan" button in the bottom right corner will become highlighted.

Step 3: Result Output

Simplot can save plots directly as BMP images or metafiles, but the resolution may not meet journal requirements. Therefore, it is recommended to export the data and plot it in Excel or other software.

1. Click on "File" -> "Save Chart Values as CSV" to export the data as a CSV file.
2. Open the CSV file in Excel, select the data, and insert a line chart to further beautify the plot.